We propose to determine the mode of binding of the HIV Nef-CD4 and Nef-MHC I interactions. NMR spectroscopy will be used to determine a solution structure of the complexes and determine the sidechains involved in direct contacts. Site-directed mutagenesis will then be utilized to produce a library of Nef variants with alanine or the wild-type amino acid residue in the mutated position. These variants will be selected for binding to the CD4 and, separately, MHC-I to determine the Nef residues that make up the functional epitope for the interaction. The binding affinities of selected Nef variants to CD4 and MHC-I will then be determined using surface plasmon resonance, fluorescence correlation spectroscopy and fluorescence polarization spectroscopy. The structure and knowledge of the functional epitope will facilitate future design of inhibitors of the interaction, which could be expanded into novel HIV therapies. [unreadable] [unreadable] [unreadable]